Human ether go-go related gene (hERG) potassium channels are of extraordinary clinical importance. hERG channels play a prominent role in the heart by generating a current that repolarizes cardiac action potentials. Mutations in the hERG gene and inhibition of hERG channels by the off-target action of prescription drugs cause a reduction in hERG current that accounts for both inherited and acquired forms of long QT syndrome (LQTS), a predisposition to cardiac arrhythmias. The disease relevance of hERG emphasizes the importance of these channels in normal physiological function. hERG channels have highly specialized gating properties (opening and closing) that optimize them for their cellular roles in the heart and specialized subunit assembly properties that also control channel gating. hERG (also known as the primary isoform, hERG1a) associates with another `alpha' subunit isoform, hERG1b. The mechanisms of subunit association are a major area of interest for understanding how heteromeric hERG1a/hERG1b channels are regulated and gated. The goal of the proposed experiments is to understand the molecular mechanisms that underlie these specializations and how they control homomeric hERG1a and heteromeric hERG1a/hERG1b channels. We will examine hERG1a and hERG1b subunit protein-protein interactions using novel fluorescence methods and protein biochemistry assays. We will test recent structures of static N- and C-terminal domain interactions within hERG1a and test how these domain interactions control assembly and dynamically rearrange during channel gating. Our approach is cutting-edge as we will use electrophysiological recordings to investigate channel conformational changes and fluorescence microscopy to study how structural interactions control channel gating and regulation. We will take advantage of non-canonical amino acid biology to engineer small probes to hERG1a and introduce metal binding sites at locations guided by recent structures and test for movements with transition metal FRET and voltage. We will also use a functional toolbox of approaches to examine the structural and functional interactions of hERG1a and hERG1b subunits and the cellular role of disease-causing mutations in human induced pluripotent stem cell-derived cardiomyocytes, which have a robust cardiac IKr current formed by hERG1a and hERG1b channel subunits. Completion of these studies will lead to a greater understanding of the basic mechanisms for homomeric hERG1a and heteromeric hERGa1a/hERG1b channel gating, insight into how intracellular domains of the channels regulate the assembly of hERG1a and hERG1b subunits and how mutations perturb these interactions. Based on our deep understanding of mechanism, we have developed and will test hERG1a polypeptides that encode hERG1a functional domains for rescue of hERG1a and hERG1b LQTS mutant channels. Our outcomes are anticipated to lead to rational biomedical strategies to counteract or enhance the loss-of-function mutations in hERG1a and hERG1b subunits that cause arrhythmias.